Sample volume, preparative

Experimentally, tire hard-sphere phase transition was observed using non-aqueous polymer lattices [79, 80]. Samples are prepared, brought into the fluid state by tumbling and tlien left to stand. Depending on particle size and concentration, colloidal crystals tlien fonn on a time scale from minutes to days. Experimentally, tliere is always some uncertainty in the actual volume fraction. Often tire concentrations are tlierefore rescaled so freezing occurs at ( )p = 0.49. The widtli of tire coexistence region agrees well witli simulations [Jd, 80]. 

A sample was prepared for analysis by diluting a 0.246-g sample to volume in a 100-mb volumetric flask. The limiting current for the sample was found to be 444 pA. Report the purity of this sample of K3be(CN)6. 

The effective use of column volume overload for preparative separations was experimentally demonstrated by Scott and Kucera [1]. These authors used a column 25 cm long, 4.6 mm I.D. packed with Partisil silica gel 10 mm particle diameter and employed n-heptane as the mobile phase. The total mass of sample injected was kept constant at 176 mg, 8 mg and 0.3 mg of benzene, naphthalene and anthracene, respectively, but the sample volumes used which contained the same mixture of solutes were 1 pi, 1 ml, 2 ml and 3 ml. The chromatograms of each separation are... 

In addition, information must be provided concerning the enrichment and clean up of the sample. If possible the sample solution prepared should be adjusted to a particular concentration, so that the application of the chosen volume gives a preliminary idea of the amounts in the chromatogram produced. 

The diameter of the column is selected from the volume of sample that is to be processed. As a rule of thumb the maximum productivity is obtained at a sample volume of 2-6% of the bed volume in preparative gel filtration on a 50-/rm chromatographic medium (Hagel et al., 1989). Thus, the required column diameter is calculated from the bed volume needed to cope with the sample volume and the column length needed to give the resolution desired. 

Sample load is primarily a concern in preparative gel filtration. In analytical applications the only precaution is to ascertain that the sample volume is sufficiently low as not to contribute to peak widths (and thus decrease the quality of the information) (Hagel, 1985). The concentration of the sample should not exceed 30 mg/ml for globular proteins or 5 mg/ml for polymers and DNA (Hagel and Janson, 1992). 

The actual loading capacity always depends on the sample composition and the separation problem. As a rule the volume of the loaded sample should not exceed 5% of the column volume. However, this recommendation is valid only for preparative runs. For analytical applications when a high resolution is needed, the volume of the injected sample should be about 1% of the total column volume or even less. For a preparative run on a 1000 X 200-mm column (bed height 60 cm), two different sample volumes were injected. If the sample volume is 0.3% of the total bed volume, the separation is more efficient... 

FIGURE 7.10 Dependence of the resolution on the sample volume. A preparative Superformance column 1000-200 (bed volume 20 liters) packed with Fractogel END BioSEC (S) (bed height 63 cm) was loaded with 60 ml (top) and 300 ml of a mixture of bovine serum albumin (5 mg/ml), ovalbumin (5 mg/ml), and cytochrome c (3 mg/ml) (bottom) (20 m/VI sodium phosphate buffer, 0.3 M NaCI, pH 7.2 flow rate 100 ml/min corresponding to 19 cm/hr). When the sample volume is 300 ml the separation efficiency for BSA and ovalbumin is similar. Thus the column can be loaded with larger sample volumes, resulting in reasonable separations. 

Volume overload employing a solution of the material in the mobile phase at a level of about 5% w/v is a recommended method of sampling for preparative columns if the system is not optimized. However, a combination of volume overload and mass overload has also been suggested as an alternative procedure by Knox (13). 

Benzene, benzene-t/i, CFCI3 and CF3CCI3 were obtained commercially and were not further purified. Solutions of c a 0.3 - 1 volume % of benzene in CFCI3 were prepared in suprasil quartz tubes of 4 mm outer diameter on a vacuum line. The samples were degassed and sealed under vacuum (< 10-4 -porr). Polycrystalline samples were prepared by rapid freezing in liquid nitrogen. The samples were irradiated at 77 K for 5 minutes at an approximate dose rate of 250 G/min. using the radiation from an X-ray tube with a W anode operated at 70 kV and 20 mA. 

A protocol must be established and followed for sample preparation, labeling, packaging, shipping, and chain-of-custody procedures. Also, the volume of the samples will be specified by the analytical laboratory depending on the analytical methods to be used and the desired sensitivity. Accordingly, principal attention will be given here to the sampling methods, preparation of the samples for analysis, and QA/QC aspects of both. 

An alternative to spray-on in the form of a rectangular area, the solid phase sample apphcation (SPSA) is suited for apphcation of especially large, nonvolatile sample volumes on preparative layers [2]. Therefore, the sample is dissolved in a suitable... 

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